Deciphering the mechanism of action of 089, a compound impairing the fungal cell cycle.

Fungal infections represent an increasingly relevant clinical problem, primarily because of the increased survival of severely immune-compromised patients. Despite the availability of active and selective drugs and of well-established prophylaxis, classical antifungals are often ineffective as resistance is frequently observed. The quest for anti-fungal drugs with novel mechanisms of action is thus important. Here we show that a new compound, 089, acts by arresting fungal cells in the G2 phase of the cell cycle through targeting of SWE1, a mechanism of action unexploited by current anti-fungal drugs. The cell cycle impairment also induces a modification of fungal cell morphology which makes fungal cells recognizable by immune cells. This new class of molecules holds promise to be a valuable source of novel antifungals, allowing the clearance of pathogenic fungi by both direct killing of the fungus and enhancing the recognition of the pathogen by the host immune system.

Insight to the binding mode of triazole RGD-peptidomimetics to integrin-rich cancer cells by NMR and molecular modeling.

The binding features of a novel class of 'click chemistry'-derived RGD mimics with integrin ligand capability were studied toward αvß3 integrin using STD-NMR techniques on intact integrin-rich ECV340 bladder cancer cell line. STD is useful to identify which moieties of the ligand are closest to the receptor in the bound state. The NMR data were integrated with competitive binding assays to the purified αvß3 receptor and were interpreted with the aid of docking calculations. The involvement of the triazole hydrogen atom in the interaction with the receptor was evinced for all compounds but 2, in agreement with docking studies showing a certain proximity between triazole and Tyr178. Moreover, the interaction of the hydroxylated ligands with the receptor was not as extended as in the compounds belonging to the corresponding series, with the exception of compound 4 having 2-aminobenzimidazole as the arginine bioisostere, in agreement with biological assay results showing reduced binding capability for the hydroxylated peptidomimetics.

Radiosynthesis and micro-SPECT analysis of triazole-based RGD integrin ligands as non-peptide molecular imaging probes for angiogenesis.

Taking advantage of click chemistry, we synthesized triazole-containing RGD peptidomimetics capable of binding to αvß3 integrin with diverse potency, and selected (125)I-labeled compounds proved to interact in vitro and in vivo with αvß3 integrin expressed by melanoma cells. Two (125)I-compounds containing either 2-aminobenzimidazole or 2-aminopyridine groups as the arginine bioisostere with the capacity to selectively bind cells of highly expressing αvß3 melanoma xenografts were found using micro-SPECT imaging studies.

Skeletal diversity from carbohydrates: use of mannose for the diversity-oriented synthesis of polyhydroxylated compounds.

The application of d-mannose as a multipurpose building block from the chiral pool enabled the diversity-oriented synthesis of an array of cyclic and bicyclic scaffolds with polyhydroxylated appendages with the aim to expand the skeletal diversity in the panorama of glycopeptidomimetic compounds.

Peptidomimetics as protein arginine deiminase 4 (PAD4) inhibitors.

The protein arginine deiminase 4 (PAD4) is a calcium-dependent enzyme, which catalyses the irreversible conversion of peptidyl-arginines into peptidyl-citrullines and plays an important role in several diseases such as in the rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, Creutzfeldt-Jacob's disease and cancer. In this study, we report the inhibition profiles and computational docking toward the PAD4 enzyme of a series of 1,2,3-triazole peptidomimetic-based derivatives incorporating the ß-phenylalanine and guanidine scaffolds. Several effective, low micromolar PAD4 inhibitors are reported in this study.

Diversity-oriented synthesis as a tool for chemical genetics.

Chemical genetics is an approach for identifying small molecules with the ability to induce a biological phenotype or to interact with a particular gene product, and it is an emerging tool for lead generation in drug discovery. Accordingly, there is a need for efficient and versatile synthetic processes capable of generating complex and diverse molecular libraries, and Diversity-Oriented Synthesis (DOS) of small molecules is the concept of choice to give access to new chemotypes with high chemical diversity. In this review, the combination of chemical genetics and diversity-oriented synthesis to identify new chemotypes as hit compounds in chemical biology and drug discovery is reported, giving an overview of basic concepts and selected case studies.

Identification of constrained peptidomimetic chemotypes as HIV protease inhibitors.

Small-molecule peptidomimetic inhibitors that already showed activity towards Secreted aspartic protease 2 as anti-Candida agents are herein presented as candidate HIV protease inhibitors. A library of 6,8-dioxa-3-azabicyclo[3.2.1]-octane peptidomimetic scaffolds was screened towards HIV protease, resulting in the identification of hit compounds possessing IC50 in the sub-micromolar range, and showing the bicyclic acetal portion as a potential transition state analogue in the interaction with catalytic aspartic acid residues.

Cyclopropane pipecolic acids as templates for linear and cyclic peptidomimetics: application in the synthesis of an Arg-Gly-Asp (RGD)-containing peptide as an αvß3 integrin ligand.

The synthesis and evaluation of substituted cyclopropane pipecolic acids (CPA) as conformationally restricted templates for linear and cyclic peptidomimetics is reported. A variety of differently substituted (poly)hydroxy- and amino-2-azabicyclo[4.1.0]heptane-1-carboxylic acids were prepared by means of the Pd-catalyzed methoxycarbonylation of suitably functionalized lactam-derived enol phosphates, followed by OH-directed cyclopropanation. CPAs were successfully introduced into a linear peptide sequence to assess the cis/trans isomerism about the pipecolic acid peptide bond, and in a cyclic peptidomimetic that bore the Arg-Gly-Asp (RGD) sequence, which displayed nanomolar activity as antagonist of the αvß3 integrin in M21 human melanoma cells. Thus, CPAs appear to be suitable for the generation of novel peptidomimetics for drug discovery.

Evaluation of efficacy, pharmacokinetics and tolerability of peptidomimetic aspartic proteinase inhibitors as cream formulation in experimental vaginal candidiasis.

OBJECTIVES: It has been previously shown that the treatment with the two protease inhibitors APG12 and APG19 confers protection in a rat model of mucosal candidiasis; in this study, we examined whether these peptidomimetic inhibitors are also effective as a cream formulation in reducing Candida albicans vaginal infection. METHODS: These efficacy studies were performed in a rat model of estrogen-dependent rat vaginitis by C. albicans on both azole-susceptible and azole-resistant C. albicans, and on both caspofungin-susceptible and caspofungin-resistant C. albicans strains. In vivo studies were also conducted in female albino rats and rabbits to obtain information about the safety, local tolerability and principal pharmacokinetics parameters of the two compounds. KEY FINDINGS AND CONCLUSIONS: Both hit compounds showed remarkable results within the 48-h range as effective inhibitors of the infection, particularly causing rapid decay of vaginal C. albicans burden. Importantly, the two compounds showed marked acceleration of fungus clearance in the rats challenged with the fluconazole-resistant as well as with the capsofungin-resistant strain of C. albicans. Both compounds showed fast elimination rates when given by the intravenous route, and poor systemic absorption after intravaginal cream administration. Test drugs were also well tolerated in 7-day local tolerability experiments in the rabbit.

A novel allergen-adjuvant conjugate suitable for specific immunotherapy of respiratory allergy.

BACKGROUND: Several approaches to find a better adjuvant, focus immunomodulation, and reduce allergenicity are under investigation to improve the efficacy and safety of specific immunotherapy. OBJECTIVE: We performed an investigation of the in vitro and in vivo effects of a purified allergen chemically conjugated to a novel 8-OH modified adenine as an adjuvant. METHODS: Purified group 2 major allergen from house dust mite chemically conjugated to 4-(6-amino-9-benzyl-8-hydroxy-9H-purin-2-ylsulfanyl)-butyric acid succinimidyl ester was analyzed by using mass spectrometry. The adduct (nDer p 2-Conj) was assayed for Toll-like receptor activation on transfected HEK293 cells, stimulation of innate cells, and effects on the functional phenotype of specific T-cell lines and clones by means of flow cytometry, real-time PCR, and expression of TH-related transcription factors. Lung cells and sera of nDer p 2-Conj-sensitized C57Bl/6 mice were studied by means of cytology, histology, real-time PCR, and ELISA. RESULTS: nDer p 2-Conj stimulated IL-12 and IFN-α production from monocytes and plasmacytoid dendritic cells, respectively, retaining the ability to trigger Toll-like receptor 7 exclusively, and expanded human allergen-specific lymphocytes with reduced ability to produce T(H)2-related cytokines and increased IFN-γ levels, as based on GATA-3/T-bet expression. In vivo adduct-sensitized mice exhibited reduced eosinophil infiltration and IL-13 expression in the airways, IFN-γ upregulation together with IgE downregulation, and an increase in allergen-specific IgG(2a) levels in sera. The conjugate exhibited reduced ability to activate human FcεRI(+) cells without inducing T(H)17 cells or autoantibodies. CONCLUSIONS: The codelivery of an allergen with a modified adenine as a conjugate inducing modulatory cytokines from innate cells redirects in vitro and in vivo pathogenic TH2 responses without eliciting harmful effects.

Insight into the structural similarity between HIV protease and secreted aspartic protease-2 and binding mode analysis of HIV-Candida albicans inhibitors.

The analysis of the structural similarity between Candida albicans Sap2 and HIV-1 aspartic proteases by molecular modeling gave insight into the common requirements for inhibition of both targets. Structure superimposition of Sap2 and HIV-1 protease confirmed the similarity between their active sites and flap regions. HIV-1 protease inhibitors herein investigated can fit the active site of Sap2, adopting very similar ligand-backbone conformations. In particular, key anchoring sites consisting of Gly85 in Sap2 and Ile50 in HIV-1 protease, both belonging to their corresponding flap regions, were found as elements of a similar binding-mode interaction. The knowledge of the molecular basis for binding to both Sap2 and HIV-1 proteases may ultimately lead to the development of single inhibitor acting on both targets.

Bicyclic peptidomimetics targeting secreted aspartic protease 2 (SAP2) from Candida albicans reveal a constrained inhibitory chemotype.

The in vitro screening of stereoisomeric bicyclic peptidomimetics towards SAP2 of Candida albicans revealed a constrained chemotype as aspartic protease inhibitor in the micromolar to nanomolar range. The results indicated that the acetal bridge may serve as a transition-state isostere, and that the right match between interactions with subsites and the orientation by hydrogen bonding with Gly85 is the main requisite for inhibitory activity. Molecular docking calculations suggested the bicyclic acetal scaffold to be capable of interacting with the two catalytic aspartic acids, thus resulting in good inhibitory activity with only two hydrophobic groups addressing the enzyme catalytic subsites.

D-proline-based peptidomimetic inhibitors of anthrax lethal factor.

In this work we reported the generation of d-proline-derived hydroxamic acids as inhibitors of anthrax lethal factor (LF), taking advantage of a pyrrolidine ring as the central scaffold and a hydroxamate group as the Zn(2+) chelating agent. The introduction of two hydrophobic groups addressing the S1' subsite and a long substrate-binding groove was conceived by overlapping the bioactive conformations of two reported LF inhibitors. Micromolar affinity of compound 38 suggested cis-3-substituted-1-sulfonamido-d-proline hydroxamic acids as a promising class of peptidomimetic inhibitors for developing novel LF inhibitors.

¹²5I-radiolabeled morpholine-containing arginine-glycine-aspartate (RGD) ligand of αvß3 integrin as a molecular imaging probe for angiogenesis.

In this paper, using a hybrid small-animal Micro SPECT/CT imaging system, we report that a new (125)I-Cilengitide-like RGD-cyclopentapeptide, containing d-morpholine-3-carboxylic acid, interacts in vivo with α(v)ß(3) integrin expressed by melanoma cells. Images clearly show that the (125)I-compound has the capacity to monitor the growth of a melanoma xenograft. Indeed, retention of the labeled ligand in the tumor mass has a good tumor/background ratio, and a significant reduction of its uptake was observed after injection of unlabeled ligand. These results suggest that the use of (125)I-labeled morpholine-based RGD-cyclopentapeptides targeting α(v)ß(3) positive tumors may play a role in future therapeutic strategies.

One-pot sequential Ti-/Cu-catalysis for tandem amidation/Ullmann-type cyclization: synthesis of model benzodiazepine(di)ones promoted by microwave irradiation.

The application of sequential Ti-/Cu-catalysis in the model one-pot synthesis of benzodiazepine(di)ones promoted by microwave irradiation demonstrates the expediency of dual catalysis in coupling-cyclization methods useful for diversity-oriented synthesis.

The TLR7 ligand 9-benzyl-2-butoxy-8-hydroxy adenine inhibits IL-17 response by eliciting IL-10 and IL-10-inducing cytokines.

This study evaluates the ability of a novel TLR7 ligand (9-benzyl-2-butoxy-8-hydroxy adenine, called SA-2) to affect IL-17 response. The SA-2 activity on the expression of IL-17A and IL-17-related molecules was evaluated in acute and chronic models of asthma as well as in in vivo and in vitro α-galactosyl ceramide (α-GalCer)-driven systems. SA-2 prepriming reduced neutrophils in bronchoalveolar lavage fluid and decreased methacoline-induced airway hyperresponsiveness in murine asthma models. These results were associated with the reduction of IL-17A (and type 2 cytokines) as well as of molecules favoring Th17 (and Th2) development in lung tissue. The IL-17A production in response to α-GalCer by spleen mononuclear cells was inhibited in vitro by the presence of SA-2. Reduced IL-17A (as well as IFN-γ and IL-13) serum levels in mice treated with α-GalCer plus SA-2 were also observed. The in vitro results indicated that IL-10 produced by B cells and IL-10-promoting molecules such as IFN-α and IL-27 by dendritic cells are the major player for SA-2-driven IL-17A (and also IFN-γ and IL-13) inhibition. The in vivo experiments with anti-cytokine receptor Abs provided evidence of an early IL-17A inhibition essentially due to IL-10 produced by resident peritoneal cells and of a delayed IL-17A inhibition sustained by IFN-α and IL-27, which in turn drive effector T cells to IL-10 production. These findings suggest that such TLR7 agonist downregulating Th17 (as well as Th2) response has to be considered a valid candidate for novel vaccine formulations in allergy.

Novel small molecules for the treatment of infections caused by Candida albicans: a patent review (2002-2010).

INTRODUCTION: The fungal pathogen Candida albicans is one of the leading causes of infections affecting immunodeficient individuals, including those HIV-infected and patients undergoing cancer therapy. Emerging problems in terms of therapeutic efficacy and drug resistance have highlighted the need to consider new therapeutic approaches, based on the exploitation of virulence factors as alternatives to conventional drug targets. AREAS COVERED: Advances in the development of anti-Candida drugs are examined in this review, as reflected by the patent literature since 2002 along with selected peer-reviewed publications. Taking into account a total of 26 patents, the discussion encompasses several therapeutic approaches, including azoles as ergosterol biosynthesis inhibitors, glucan and chitin synthase inhibitors, and secreted aspartyl protease inhibitors. EXPERT OPINION: New analogs of existing drugs are being developed as broad spectrum antifungals to improve efficacy and circumvent drug resistance. Also, candidate drugs targeting new virulence factors are promising to overcome limitations due to poor efficacy and the rising of drug resistance observed for several available drugs. Efforts for the discovery and development of antifungal agents should be equivalent to other therapeutic areas, and advances in the generation of therapeutic agents with fungus-specific mechanisms of action are of highest priority.

A new family of cinchona-derived amino phosphine precatalysts: application to the highly enantio- and diastereoselective silver-catalyzed isocyanoacetate aldol reaction.

A new class of readily accessible chiral amino-phosphine precatalysts derived from 9-amino(9-deoxy) epicinchona alkaloids has been developed. In combination with Ag(I) salts, these amino-phosphines performed as effective cooperative Brønsted base/Lewis acid catalysts in the asymmetric aldol reaction of isocyanoacetate nucleophiles. Under optimal conditions, high diastereoselectivities (up to 98%) and enantioselectivities (up to 98%) were obtained.

Chemical genetics approach to identify new small molecule modulators of cell growth by phenotypic screening of Saccharomyces cerevisiae strains with a library of morpholine-derived compounds.

A chemical genetics approach has been applied in the screening of yeast deletants strains with a pool of morpholine-derived compounds in order to identify candidate small molecules able to produce phenotypic effects on yeast cells. The analysis of the effects of structurally diverse molecules towards cell growth rate in both exponential and stationary phases provides a tool to select candidate compounds for subsequent assays to identify new chemical entities as chemical probes for drug discovery.

Click-chemistry-derived triazole ligands of arginine-glycine-aspartate (RGD) integrins with a broad capacity to inhibit adhesion of melanoma cells and both in vitro and in vivo angiogenesis.

A click chemistry approach was applied for the discovery of triazole-based arginine-glycine-aspartate (RGD) mimetics by Cu(I)-catalyzed 1,3-dipolar alkyne-azide coupling reaction, which showed binding affinity properties toward α(v)ß(3)/α(v)ß(5) integrins. Biological assays showed compound 18 capable of binding α(v)ß(3) integrin with nanomolar affinity according to a two-sites model, and molecular modeling studies revealed a peculiar π-stacking interaction between the triazole ring and Tyr178 side chain. Accordingly, compound 18 inhibited the adhesion of integrin-expressing human melanoma cells to RGD-containing proteins of the extracellular matrix, such as vitronectin, fibronectin, and osteopontin, and also angiogenesis in in vitro and in vivo experimental models. The relevant biological effects exerted by compound 18 suggest its potential application as an antiangiogenic agent in the diagnosis and therapy of tumors where α(v)ß(3) integrin expression is up-regulated.